Activating pyruvate kinase r and mutants thereof

ABSTRACT

The disclosure provides chemical compounds useful as activators of PKR. PKR activating compounds are useful in the treatment of disease and disorders associated with modulation of PKR and/or PKM2, such as pyruvate kinase deficiency (PKD).

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No. 16/575,456, filed Sep. 19, 2019, which claims the benefit of U.S. Provisional Application No. 62/733,546, filed on Sep. 19, 2018, each of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This disclosure relates to novel chemical compositions for activating the pyruvate kinase enzyme, useful in the treatment of pyruvate kinase-related medical conditions (e.g., pyruvate kinase deficiency).

BACKGROUND

Pyruvate Kinase (PK) converts phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP) to pyruvate and adenosine triphosphate (ATP), respectively, which is the final step in glycolysis. In humans, four PK isoforms are expressed by two structural genes. The PKLR gene encodes PKR and PKL, tissue specific isoforms expressed in erythroid cells and liver cells, respectively. The PKM gene codes for isoforms PKM1, expressed in brain and skeletal muscle, and PKM2 (M2-type pyruvate kinase), expressed in fetal and most adult tissues except erythroid cells.

Mutations in the PKLR gene can lead to pyruvate kinase deficiency (PKD), an autosomal recessive disorder, which is the most frequent enzymatic defect of the glycolytic pathway in erythrocytes. Over 200 different mutations have been identified on the structural PKLR gene. Generally, most PKD patients are heterozygous with two different mutant alleles, but homozygous mutations have also been described. Clinical symptoms of PKD vary considerably from mild to severe anemia. Mutations can reduce PK enzymatic activity or decrease PK protein stability. Pathological manifestations are usually observed when enzyme activity falls below 25% normal PK activity, and severe disease has been associated with a high degree of reticulocytosis. Although the global incidence of PKD is unknown, it has been estimated at 51 cases per million in North America. Heterogenous genetic mutations in PKR can lead to dysregulation of its catalytic activity. These mutations represent wide range genetic lesions that include deletional and transcriptional or translational abnormalities, including missense mutation in the coding region that can affect conserved residues within domains that are structurally important for optimal catalytic function of PKR.

Currently, there is no definitive treatment for severe PKD. Although splenectomy can be clinically useful in patients with severe disease, in some cases, allogeneic hematopoietic transplantation is required. In these patients, hematopoietic stem cell (HSC) gene therapy might be a good and more effective treatment. Gene therapy strategies for PKD have been addressed in animal models demonstrating that introduction of the correct version of the human PKLR gene into hematopoietic stem cells using retroviral vectors alleviates the disease. Although bone marrow transplant (BMT) or gene therapy strategies would be definitive treatments of the disease, they have yet to be realized clinically, and important adverse effects are associated with both approaches.

There remains a need for strategies to improve the treatment of diseases related to PKR, such as PKD, including the discovery and development of PKR activating small molecules. PKR exists in both a dimeric and tetrameric state, but functions most efficiently as a tetramer. Small molecules have been shown to be capable of shifting the equilibrium of PKR to the tetrameric (most active) form, providing a mechanistic rationale for their use as therapy for PKD-associated hemolytic anemia. Thus, there is a need for PKR activating compounds, useful for treating diseases and disorders associated with modulation of PKR and/or PKM2.

SUMMARY

The disclosure relates in part to compounds and compositions useful for activating PKR, including the discovery of a novel compound active with wild type (wt) PKR and/or mutant isozyme forms of PKR. For example, compounds and methods provided herein can be used for activating wild type PKR isozyme and/or a mutant PKR isozyme. Examples of mutant PKR isozyme include G332S, G364D, T384M, G37E, R479H, R479K, R486W, R532W, R510Q, 190N, and R490W. The invention is based in part on the discovery of a novel compound demonstrating activation of (wt) PKR and the G332S and R510Q mutant forms of PKR.

A PKR activator can be a compound identified as a PKR Activating Compound, defined herein as having one or more of the following characteristics when tested according to the Luminescence Assay Protocol of Example 3 below: (1) an AC₅₀ value of less than 40 μM; (2) a maximum % Fold (MAX % Fold) value of greater than 75%; and/or (3) a % Fold value at 1.54 μM compound concentration (% Fold@1.54 μM) of at least 75%. In some embodiments, the PKR Activating Compound has: (1) an AC₅₀ value of less than 0.1 μM, 0.1-1.0 μM, or 1.01-40 μM; (2) a MAX % Fold of 75%-250%, 251-500%, or 75%-500%; and/or (3) a % Fold@1.54 μM of 75%-250%, 251-500%, or 75%-500%. In some embodiments, a PKR Activating Compound has (1) an AC₅₀ value of less than 1.0 μM; (2) a MAX % Fold of 75%-500%; and/or (3) a % Fold@1.54 μM of 75%-500%. In some embodiments, the Luminescence Assay Protocol of Example 3 is performed with wild type (wt) PKR, G332S mutant form of PKR or R510Q mutant form of PKR. In some embodiments, a PKR Activating Compound can be the compound (2R)-2-hydroxy-2-phenyl-1-[5-(pyridine-2-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]ethan-1-one (Compound 1):

or a pharmaceutically acceptable salt thereof, having (1) an AC₅₀ value of less than 1.0 μM; (2) a MAX % Fold of 75%-500%; and/or (3) a % Fold@1.54 μM of 75%-500%.

A method for treating pyruvate kinase deficiency (PKD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of: (a) Compound 1 or a pharmaceutically acceptable salt thereof; (b) a composition comprising Compound 1 or a salt thereof and a carrier; or (c) a pharmaceutical composition comprising Compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, to thereby treat PKD in the subject. The method can include or result in activating one or more isozymes of pyruvate kinase. One or more isozymes of pyruvate kinase can be selected from PKR, PKM2, and PKL; the method can also include or result in activating a mutant PKR isozyme.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows an exemplary dose-response curve for compounds disclosed herein. Dose-response curves may be generated using the standard four parameter fit algorithm of ActivityBase XE Runner to determine MAX % Fold, MIN % Fold, slope and AC₅₀. MAX % Fold is the highest % fold increase observed at any concentration of compound, and MIN % Fold is the lowest % fold increase observed at any concentration of compound. The AC₅₀ value for a compound is the concentration (μM) corresponding to the midway between the maximum and minimum values of the four parameter logistic curve fit (i.e., at which the % fold increase along the four parameter logistic curve fit is halfway between MAX % Fold and MIN % Fold (% Fold Midpoint). Another useful parameter for evaluating compounds of this disclosure is % Fold@1.54 μM, which is the % fold increase at a compound concentration of 1.5 μM (e.g., 1.54 μM). X-axis and y-axis not necessarily to scale.

DETAILED DESCRIPTION

In some embodiments, the present disclosure provides a PKR Activating Compound which is Compound 1:

or a pharmaceutically acceptable salt thereof. In some embodiments, the PKR Activating Compound is (2R)-2-hydroxy-2-phenyl-1-[5-(pyridine-2-sulfonyl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl]ethan-1-one. Compound 1 is useful for the activation of PKR and mutant isozyme forms thereof.

The present disclosure also provides compositions (e.g. pharmaceutical compositions) comprising Compound 1. The pharmaceutical compositions provided herewith may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, and tablets (e.g., in unit dosage forms). In some embodiments, Compound 1 is formulated for oral administering to the subject at a dose of about 10 mg to about 1000 mg, e.g., as a capsule or tablet containing about 50 mg-about 100 mg of Compound 1 and pharmaceutically acceptable carriers and/or excipients. Lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient's disposition to the disease, condition or symptoms, and the judgment of the treating physician. Pharmaceutical compositions of Compound 1 may comprise one or more additional excipients and/or carriers, with Compound 1 present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% by weight of the pharmaceutical composition.

Compounds and compositions described herein are activators of wild type PKR and certain PKR mutants having lower activities compared to the wild type. Such mutations in PKR can affect enzyme activity (catalytic efficiency), regulatory properties and/or thermostability of the enzyme. One example of a PKR mutation is G332S. Another example of a PKR mutation is R510Q. Methods of treatment (e.g., by activating wild type PKR) can comprise administering to a subject in need thereof a therapeutically effective amount of (1) a compound disclosed herein or a pharmaceutically acceptable salt thereof; (2) a pharmaceutical composition comprising a compound disclosed herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The pharmaceutical compositions may be orally administered in any orally acceptable dosage form. In some embodiments, to increase the lifetime of red blood cells, a compound, composition or pharmaceutical composition described herein is added directly to whole blood or packed cells extracorporeally or provided to the subject (e.g., the patient) directly. The compositions described herein can modulate (e.g., activate) PKR. Accordingly, a patient and/or subject can be selected for treatment using a compound described herein by first evaluating the patient and/or subject to determine whether the subject is in need of modulation (e.g., activation) of PKR, and if the subject is determined to be in need of modulation of PKR, then administering to the subject a composition described herein.

Compound 1 can activate wild type PKR and/or mutant PKRs. Some examples of the mutant PKRs include G332S, G364D, T384M, G37E, R479H, R479K, R486W, R532W, R510Q, I90N, and R490W. Accordingly, a patient and/or subject can be selected for treatment using Compound 1 by first evaluating the patient and/or subject to determine whether the subject carries a mutation in PKR (for example, one of the mutations as described herein), and if the subject is determined to be carrying a mutation in PKR thus is in need of activation of the activity of the mutant PKR, then administering to the subject a therapeutically effective amount of Compound 1. A subject can be evaluated as carrying a mutation in PKR using methods known in the art. The subject can also be monitored, for example, subsequent to administration of Compound 1.

A patient can be monitored for evaluation of certain PK/PD parameters of Compound 1 such as levels of Compound 1, levels of 2,3-DPG, or levels of ATP.

In some embodiments, the disclosure relates to a method of treating a patient diagnosed with a pyruvate kinase-related medical condition, the method comprising administering to the patient in need thereof a therapeutically effective amount of a PKR Activating Compound having a maximum % Fold (MAX % Fold) value of greater than 75%. In some embodiments, the disclosure relates to a method of treating a patient diagnosed with a pyruvate kinase-related medical condition, the method comprising administering to the patient in need thereof a therapeutically effective amount of a PKR Activating Compound having a % Fold value at 1.54 μM compound concentration (% Fold@ 1.54 μM) of at least 75%.

In one aspect, the present invention provides a method of treating a subject, the method comprising: administering to the subject (2R)-2-Hydroxy-2-phenyl-1-(5-(pyridin-2-ylsulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)ethan-1-one (Compound 1); and acquiring a value for the level of Compound 1, the level of 2,3-diphosphoglycerate (2,3-DPG), the level of adenosine triphosphate (ATP), or the activity of PKR in the subject, to thereby evaluate the subject. The value for the level of Compound 1 can be acquired by analyzing the plasma concentration of Compound 1. In some embodiments, the level of 2,3-DPG is acquired by analyzing the blood concentration of 2,3-DPG. The level of ATP can be acquired by analyzing the blood concentration of ATP. The activity of PKR can be acquired by analyzing the blood concentration of a ¹³C-label in the blood. For example, ¹³C-labeled glucose is administered to a subject, and incorporated into certain glycolytic intermediates in the blood.

A method of treatment can comprise administering an amount of Compound 1 sufficient to provide a blood concentration of 2,3-DPG that is reduced by at least 15% relative to the reference standard (e.g., from about 15% to about 60%). In some embodiments, the blood concentration of 2,3-DPG is reduced by at least about 15%, by at least about 20%, by at least about 25%, by at least about 30%, by at least about 35%, by at least about 40%, by at least about 45%, by at least about 50%, by at least about 55%, by at least about 60%. In some embodiments, the analysis is performed by sample analysis of bodily fluid, such as blood, by e.g., mass spectroscopy, e.g. LC-MS.

The present disclosure enables one of skill in the relevant art to make and use the inventions provided herein in accordance with multiple and varied embodiments. Various alterations, modifications, and improvements of the present disclosure that readily occur to those skilled in the art, including certain alterations, modifications, substitutions, and improvements are also part of this disclosure. Accordingly, the foregoing description and drawings are by way of example to illustrate the discoveries provided herein.

EXAMPLES Example 1: Synthesis of (2R)-2-Hydroxy-2-phenyl-1-(5-(pyridin-2-ylsulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)ethan-1-one Intermediate A: tert-Butyl 5-(pyridin-2-ylsulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate

To a suspension of tert-butyl 3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate, HCl (1.10 g, 4.46 mmol) in dichloromethane (25 mL) at room temperature was added TEA (1.49 mL, 10.7 mmol) followed by pyridine-2-sulfonyl chloride (0.950 g, 5.35 mmol). The reaction mixture was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure and the residue was taken up in EtOAc (50 mL). The organic mixture was sequentially washed with water (50 mL) and saturated aqueous sodium chloride solution (50 mL). The organic layer was collected, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified by silica gel column chromatography (0% EtOAc/hexane for 5 min then 0-75% EtOAc/hexane for 30 min) to afford tert-butyl 5-(pyridin-2-ylsulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (0.57 g, 1.622 mmol, 36.4% yield). LCMS (m/z, M+H+) 351.9, 1H-NMR (300 MHz, CDCl3): δ ppm 8.69 (m, 1H), 8.00-7.88 (m, 2H), 7.49 (m, 1H), 4.33 (m, 4H), 4.05 (m, 4H), 1.45 (s, 9H).

Intermediate B: 2-(Pyridin-2-ylsulfonyl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole, HCl

To a solution of tert-butyl 5-(pyridin-2-ylsulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (0.570 g, 1.62 mmol) in dichloromethane (4.96 ml) was added 4 M HCl/dioxane (17.6 ml). The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure then triturated with ether. The white solid was filtered off to give 2-(pyridin-2-ylsulfonyl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole, HCl (0.547 g, 1.90 mmol, quantitative yield) that was used in the next step without further purification. LCMS (m/z, M+H+) 252.0, 1H-NMR (300 MHz, DMSO-d6): δ ppm 9.99 (br m, 2H), 8.75 (m, 1H), 8.13 (m, 1H), 7.97 (m, 1H), 7.71 (m 1H), 4.24 (m, 4H), 3.89 (m, 4H).

(R)-2-Hydroxy-2-phenyl-1-(5-(pyridin-2-ylsulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)ethan-1-one

To a suspension of 2-(pyridin-2-ylsulfonyl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole, HCl (0.547 g, 1.90 mmol) in dichloromethane (12.6 mL) was added (R)-2-hydroxy-2-phenylacetic acid (0.241 g, 1.58 mmol), BOP (0.771 g, 1.74 mmol) and DIEA (0.830 ml, 4.75 mmol). The reaction mixture was stirred at room temperature for 2 hours. Ethyl acetate (50 mL) was added and the organic phase was washed with saturated aqueous sodium hydrogen carbonate solution (50 mL) and saturated aqueous sodium chloride solution (50 mL). The organic phase was dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel column chromatography on (gradient of 0-10% MeOH/dichloromethane) to afford (R)-2-hydroxy-2-phenyl-1-(5-(pyridin-2-ylsulfonyl)-5,6-dihydropyrrolo[3,4-c]pyrrol-2(1H,3H,4H)-yl)ethanone (0.353 g, 0.916 mmol, 57.8% yield). LCMS (m/z, M+H+) 385.9, 1H-NMR (300 MHz, DMSO-d6): δ ppm 8.69 (m, 1H), 8.07 (m, 1H), 7.91 (m, 1H), 7.68-7.64 (m, 1H), 7.33-7.26 (m, 5H), 5.64 (d, J=6.0 Hz, 1H), 5.14 (d, J=6.3 Hz, 1H), 4.30-3.88 (m, 8H).

Example 2. 1-(5-[[4-(Difluoromethoxy)benzene]sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-2-hydroxy-2-(pyridin-3-yl)ethan-1-one

Into a 8-mL vial (1 atm), was placed a mixture of 2-hydroxy-2-(pyridin-3-yl)acetic acid (39.8 mg, 0.26 mmol, 1.30 equiv), N,N-dimethylformamide (1.5 mL), 2-((4-(difluoromethoxy)phenyl) sulfonyl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrolehydrochloride (70.6 mg, 0.20 mmol, 1.00 equiv), DIEA (65 mg, 0.50 mmol, 2.50 equiv) and HATU (91 mg, 0.24 mmol, 1.20 equiv). The resulting solution was stirred for 16 h at room temperature. The reaction solution was diluted with 50 mL of ethyl acetate, washed with 4×10 mL of brine, dried over anhydrous sodium sulfate and concentrated under vacuum. The crude product was purified by prep-TLC. This resulted in 1-(5-[[4-(Difluoromethoxy)benzene]sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-2-hydroxy-2-(pyridin-3-yl)ethan-1-one as an off-white solid. ¹H NMR (300 MHz, CDCl₃): δ ppm 8.66 (d, J=4.8 Hz, 1H), 8.65-7.87 (m, 2H), 7.51-7.48 (m, 1H), 7.48-7.31 (m, 5H), 5.01 (s, 1H), 4.32-4.15 (m, 7H), 3.63 (d, J=12.3 Hz, 1H). LCMS m/z: 452 [M+H]⁺.

Compound 1 in Table 1 below was prepared according to the procedure outlined above for Example 2, using the appropriate synthetic precursors.

TABLE 1 Structure LCMS Example Name (m/z) ¹H-NMR Compound 1

386 (300 MHz, CDCl₃): δ ppm 8.66 (d, J = 4.8 Hz, 1H), 8.65-7.87 (m, 2H), 7.51- 7.48 (m, 1H), 7.48-7.31 (m, 5H), 5.01 (s, 1H), 4.32-4.15 (m, 7H), 3.63 (d, J = 12.3 Hz, 1H)

Example 3. Luminescence Biochemical Assay

In some embodiments, a PKR Activating Compound refers to a compound having one or more characteristics when tested according to the following Luminescence Assay Protocol of Example 3 performed with wild type (wt) PKR and/or any one or more of G332S mutant form of PKR or R510Q mutant form of PKR: (1) an AC₅₀ value of less than 40 μM (e.g., compounds with AC₅₀ values of “+”, “++”, or “+++” in Table 2); (2) a maximum % Fold (MAX % Fold) value of greater than 75%; and/or (3) a % Fold value at 1.54 μM compound concentration (% Fold@1.54 μM) of at least 75%. In some embodiments, a PKR Activating Compound can have: (1) an AC₅₀ value of less than 0.1 μM (e.g., compounds with AC₅₀ values of “+++” in Table 2), 0.1-1.0 μM (e.g., compounds with AC₅₀ values of “++” in Table 2), or 1.01-40 μM (e.g., compounds with AC₅₀ values of “+” in Table 2); (2) a MAX % Fold of 75%-250%, 250-500%, or 75%-500%; and/or (3) a % Fold@1.54 μM of 75%-250%, 250-500%, or 75%-500%. In some embodiments, a PKR Activating Compound has (1) an AC₅₀ value of less than 1.0 μM; (2) a MAX % Fold of 75%-500%; and/or (3) a % Fold@1.54 μM of 75%-500%.

The phosphorylation of Adenosine-5′-diphosphate (ADP) by various mutants of PKR was determined by the Kinase Glo Plus Assay (Promega) in the presence or absence of FBP [D-Fructose-1,6-diphosphate; BOC Sciences, CAS: 81028-91-3] as follows. Unless otherwise indicated, all reagents were purchased from Sigma-Aldrich. All reagents were prepared in buffer containing 50 mM Tris-HCl, 100 mM KCl, 5 mM MgCl₂, and 0.01% Triton X100, 0.03% BSA, and 1 mM DTT. Enzyme and PEP [Phospho(enol) pyruvic acid] were added at 2× to all wells of an assay-ready plate containing serial dilutions of test compounds or DMSO vehicle. Final enzyme concentrations for PKR(wt), PKR(R510Q), and PKR(G332S) were 0.8 nM, 0.8 nM, and 10 nM respectively. Final PEP concentration was 100 μM. The Enzyme/PEP mixture was incubated with compounds for 30 minutes at RT before the assay was initiated with the addition of 2×ADP [Adenosine-5′-diphosphate] and KinaseGloPlus. Final concentration of ADP was 100 μM. Final concentration of KinaseGloPlus was 12.5%. For assays containing FBP, that reagent is added at 30 μM upon reaction initiation. Reactions were allowed to progress for 45 minutes at RT until luminescence was recorded by the BMG PHERAstar FS Multilabel Reader. All compounds were tested in triplicate at concentrations ranging from 42.5 μM to 2.2 nM in 0.83% DMSO.

Luminescence values were converted to % Fold increase by normalizing to the average of the DMSO control and multiplying by 100. Max, min, slope and AC₅₀ were determined by the standard four parameter fit algorithm of ActivityBase XE Runner. Compounds were evaluated with three parameters—AC₅₀, MAX % Fold, and % Fold@1.54 μM (FIG. 1). The AC₅₀ value for a compound is the concentration (μM) corresponding to the midway between the maximum and minimum values of the four parameter logistic curve fit (i.e., at which the % fold increase along the four parameter logistic curve fit is halfway between MAX % Fold and MIN % Fold (% Fold Midpoint)), MAX % Fold is the highest fold increase observed at any concentration of compound, and % Fold@1.54 μM is the fold increase at a compound concentration of 1.54 μM. The parameter % Fold@1.54 μM was selected to capture elements of both the AC₅₀ and MAX % Fold and to provide a ranking based on both potency and effect. The compound concentration of 1.54 μM was chosen as one that can optimally differentiate the set of compounds based on the range of activities observed.

As set forth in Table 2 below, AC₅₀ values (columns A, D, G) are defined as follows: ≤0.1 μM (+++); >0.1 μM and ≤1.0 μM (++); >1.0 μM and ≤40 μM (+); >40 μM (0). Max % FOLD values (columns B, E, H) are defined as follows: ≤75% (+); >75% and ≤250% (++); >250% and ≤500% (+++). % Fold@1.54 μM values (columns C, F, J) are defined as follows: ≤75% (+); >75% and ≤250% (++); >250% and ≤500% (+++).

TABLE 2 PKRG332S PKRR510Q WT Conditions¹ Conditions¹ Conditions¹ Compound A B C D E F G H J 1 ++ ++ ++ ++ +++ ++ +++ ++ ++ ¹A - AC50 LUM KGP FBP AC₅₀ μM gmean; B - AC50 LUM KGP FBP MAX % FOLD mean; C - AC50 LUM KGP FBP % Fold@ 1.54 μM mean D - AC50 LUM KGP woFBP AC₅₀ μM gmean; E - AC50 LUM KGP woFBP MAX % FOLD mean; F - AC50 LUM KGP woFBP % Fold@ 1.54 μM mean G - AC50 LUM KGP woFBP AC₅₀ μM gmean; H - AC50 LUM KGP woFBP MAX % FOLD mean; J - AC50 LUM KGP woFBP % Fold@ 1.54 μM mean

TABLE 3 Biological Data of Additional Compounds PKR PKR G332S R510Q wt Conditions¹ Compound Structure A D G 23

0 0 24

+ 25

+ + 26

+ 27

+ 28

+ 29

+ 30

+ + + ¹A - AC50 LUM KGP FBP AC₅₀ μM gmean; D - AC50 LUM KGP woFBP AC₅₀ μM gmean. G - AC50 LUM KGP woFBP AC₅₀ μM gmean; 

We claim:
 1. A method of making Compound 1 comprising the step of reacting 2-(Pyridin-2-ylsulfonyl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole (Intermediate B) with (R)-2-hydroxy-2-phenylacetic acid in a reaction mixture to obtain Compound 1:


2. The method of claim 1, further comprising the step of preparing a HCl salt of Intermediate B by reacting tert-butyl 5-(pyridin-2-ylsulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (Intermediate A) in a reaction mixture comprising HCl/dioxane and dichloromethane to obtain the 2-(pyridin-2-ylsulfonyl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole HCl.
 3. The method of claim 2, further comprising the step of preparing Intermediate A by reacting tert-butyl 3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate HCl in a reaction mixture comprising pyridine-2-sulfonyl chloride and TEA to obtain tert-butyl 5-(pyridin-2-ylsulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate HCl.
 4. A compound selected from the group consisting of:

or a salt thereof.
 5. The compound of claim 4, wherein the compound is:

or a salt thereof.
 6. The compound of claim 4, wherein the compound is:

or a salt thereof.
 7. The compound of claim 6, comprising the hydrochlorate salt of Intermediate B.
 8. A composition comprising the compound tert-Butyl 5-(pyridin-2-ylsulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (Intermediate A) or a salt thereof.
 9. The composition of claim 8, wherein the composition is a reaction mixture comprising tert-butyl 3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate or a salt thereof, pyridine-2-sulfonyl chloride, TEA and tert-butyl 5-(pyridine-2-ylsulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (Intermediate A) or a salt thereof.
 10. The composition of claim 8, wherein the composition is a reaction mixture comprising tert-butyl 5-(pyridin-2-ylsulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (Intermediate A) or a salt thereof, HCl/dioxane, dichloromethane and 2-(pyridin-2-ylsulfonyl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole (Intermediate B) or a salt thereof.
 11. A composition comprising the compound 2-(Pyridin-2-ylsulfonyl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole (Intermediate B) or a salt thereof.
 12. The composition of claim 12, wherein the composition is a reaction mixture comprising 2-(Pyridin-2-ylsulfonyl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole (Intermediate B) or a salt thereof, (R)-2-hydroxy-2-phenylacetic acid and Compound 1 